This invention relates to the use of a glass support derivatized with 2-(4-chlorosulfonylphenyl) ethyl trimethoxy silane (silyl CSP), N-trimethoxysilylpropyl-N,N,N-trimethyl ammonium chloride (silyl TMA) or a combination thereof, in the sequencing of peptides or proteins.
The primary sequence of amino acids in a peptide or protein is commonly determined by a stepwise chemical degradation process in which amino acids are removed one-by-one from one end of the peptide, and identified. In the Edman degradation, the N-terminal amino acid of the peptide is coupled to phenylisothiocyanate to form the phenylthiocarbamyl (PTC) derivative of the peptide. The PTC peptide is then treated with strong acid, cyclizing the PTC peptide at the first petide bond and releasing the N-terminal amino acid as the anilinothiozolinone (ATZ) derivative. The ATZ amino acid, which is highly unstable, is extracted and converted into the more stable phenylthiohydantoin (PTH) derivative and identified by chromatography. The residual peptide is then subjected to further stepwise degradation.
Automatic protein sequencers in which the Edman degradation reactions are carried out in a film on the inside surface of spinning cup are known. Edman and Begg, Eur. J. Biochem., 1:80 (1967); Penhast, U.S. Pat. No. 3,725,010. A quaternary ammonium salt, 1,5-dimethyl-1,5-diazundecamethylene polymethobromide (Polybrene) has been used as a sample carrier.
Schroeder, Meth. Enzymol., 11:445 (1967) and Jentsch, Jr., Proc. First Int.varies.l Conf. on Meth. in Protein Sequence Anal 193 (1975) modified the liquid phase Edman degradation by first non-chemically depositing the protein or peptide onto a paper strip.
Laursen, Meth. Enzymnol., 25:344 (1972) covalently attached peptides to an insoluble resin prior to sequencing. Wachter, et al., FEBS Lett., 35: 97 (1973) immobilized the peptides on controlled pore glass beads derivatized with 3-aminopropyltriethoxysilane. The resulting aminopropyl glass binds to free carboxyl groups of the peptides Dreyer, U.S. Pat. No. 4,065,412 favors immobilizing the peptides, either by covalent linkage or by adsorption, onto a macroporous reaction support surface of polystyrene or glass.
Hood, U.S. Pat. Nos. 4,704,256 and 4,603,114 teaches embedding the sample in a permeable solid matrix formed as a thin film on a support such as a glass fiber sheet. The matrix is preferably a polymeric quaternary ammonium salt, since the positively charged quaternary ammonium groups interact strongly with the negatively charged glass surface.
FIGS. 17A, 17B and 17C in the Hood patents illustrate three approaches to improving sample retention by immobilizing the protein or peptide. FIG. 17A shows covalent attachment to derivatized glass, as in Wachter. FIG. 17B shows physical adsorption of the sample to the support, as in Schroeder. FIG. 17C illustrates embedding the sample in a matrix covering the glass, as in Hood.
Saunders, U.S. Pat. No. 3,987,058 discloses the use of sulfonated aralkyl silicas as cation exchangers. The sodium salt of sulfobenzylsilica was used to separate nitrosoproline from proline and other components of cured meat samples. There is no discussion of protein sequencing.
Glajch, U.S. Pat. No. 4,705,725 relates particularly to support structures comprising silica to which is covalently attached a monofunctional saline-containing at least two sterically protecting groups attached to the silicon atom of the silane. For use in peptide sequencing, this silane is substituted with a quaternary amino group so as to interact with peptides in a manner similar to that of Polybrene. For use in cation exchange chromatography, this silane is substituted with a --(CH.sub.2).sub.3 --C.sub.6 H.sub.4 --SO.sub.3 H group.
Silanes have been used to couple antigens or antibodies to glass. Weetall, U.S. Pat. No. 3,652,761.